Treatment of HER2+ breast cancer with the targeted therapeutic lapatinib has shown promising results, but faces the major obstacles of de novo and acquired resistance in clinical use. Much of this resistance can be attributed to intratumoral heterogeneity giving rise to drug resistant cell populations. An important factor of intratumoral heterogeneity is the spatial heterogeneity of cancer cells within the tumor mass, which results in differential contact with extra cellular matrix (ECM) proteins, stromal cells, growth factors, and other signaling molecules within the tumor microenvironment. This can variably alter the intracellular signaling network of cancer cells, modulating cellular plasticity and drug response. The goal of this project is to assess what effect cellular interactions with the microenvironment have on lapatinib resistance in HER2+ breast cancer. To accomplish this I am utilizing MicroEnvironment MicroArrays (MEMAs), which consist of combinations of functional ECMs, growth factors, and cytokines printed onto a solid surface, allowing for the simultaneous interrogation of thousands of unique microenvironments in a single assay. I have grown HER2+ breast cancer cells on MEMA spots, treated with lapatinib, and measured functional response to each protein combination by immunofluorescent assay and high throughput image acquisition and analysis. By focusing on markers of proliferation, apoptosis, and cellular subtype differentiation, I have identified several protein combinations that confer resistance to lapatinib In five luminal-like HER2+ cell lines I have identified HGF and NRG-1 as growth factors capable of providing resistance to the anti-proliferative effects of lapatinib. Additionally, I have identiied several juxtacrine signaling molecules that can modulate this growth factor mediated resistance, and have discovered that their effects correlate with the mammary tissue compartment these proteins normally localize to. Proteins such as desmoglein 2 or E-cadherin that are normally found in the luminal compartment of mammary ducts are observed to enhance NRG-1 mediated resistance to lapatinib, while ECM proteins of the basement membrane and stroma tend to diminish this resistance. HGF and NRG-1, as well as desmoglein 2 and E-cadherin, were all previously found to be important in the progression of breast cancer, but their effect in combination is a novel finding made possible by the use of MEMAs. The overall hypothesis of this project is that juxtacrine signaling molecules of the mammary duct lumen promote a luminal cellular phenotype that drives expression of key growth factor receptors, sensitizing cancer cells to growth factor mediated drug resistance. Additionally, proteins of the basement membrane and stroma can antagonize this rescue. To follow up on these findings I intend to utilize RNAseq analysis to inform a probabilistic computational model of network signaling to determine what pathways are enhanced by the proteins both alone and in combination. siRNA knockdown of identified pathway nodes can then determine which interactions can be targeted to restore drug sensitivity.